Immortalized hypothalamic neuronal cell lines

ABSTRACT

The present invention is directed to a method of preparing cell lines of hypothalamic origin. The method involves infecting fetal hypothalamic cells with a retroviral vector harbouring a viral oncogene, preferably SV-40 large T antigen, followed by selection and cloning. A plurality of cell lines have been prepared which express a variety of neuronal markers. The cell lines of the present invention are useful in the development of experimental models and in the treatment of disease.

This application claims priority from U.S. application Nos. 60/376,879,filed May 2, 2002, and 60/377,231, filed May 3, 2002, which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention is directed to immortalized, hypothalamic neuronalcell lines to methods of making same and to methods and uses thereof.

BACKGROUND OF THE INVENTION

The mammalian central nervous system is the most complex organ systemwith an estimated 10¹² different neurons that are consideredbiochemically and phenotypically unique (1). Neuronal cell types aretypically defined by their individual patterns of neurotransmitterhormone secretion, expression of cell surface molecules and receptors,signaling properties, and morphology. The hypothalamus is a criticalpart of the brain that is considered by many to be the life controlsystem in the body. It comprises a complex array of distinct neuronalphenotypes, each expressing a specific complement of neuropeptides,neurotransmitters and receptors. The neuroendocrine hypothalamusconsists of a complex array of distinct neuronal phenotypes, eachexpressing a specific complement of neuropeptides, neurotransmitters andreceptors (1). Many of our vital needs, such as those for growth,reproduction, nutrition (e.g. food and drink), sleep, and stressresponses, depend on hormonal balance or homeostasis, which iscontrolled by both external and internal stimuli at the hypothalamiclevel. The hypothalamus produces a number of peptide releasing factorsand neuropeptides that in turn control the activities of the pituitaryand other organs in the body. Knowledge of the control mechanisms ofunique peptineurgic neurons from the hypothalamus is critical before wecan understand how the brain achieves its diverse central control ofbasic physiology. Numerous studies have been undertaken to map theafferent connections between distinct hypothalamic neurons utilizingmethodology such as double-, and recently, triple-basedimmunocytochemistry and in-situ hybridization. (2-5). These studies areuseful to generate an emerging picture of the potential cellularcommunication within the hypothalamus, but are not comprehensive and donot address the mechanisms involved in gene regulation and cellularsignaling.

The lack of appropriate cell models is currently hindering thesestudies, as analysis of brain slices or whole animal experimentationyields limited, and often conflicting, mechanistic data. Non-transformedprimary hypothalamic cultures are difficult to maintain, have a shortlife-span and represent a heterogeneous neuronal and glial cellpopulation, usually with a minimal number of healthy peptide-secretingneurons.

The complexity of the hypothalamus, due to numerous cells harbouringunique characteristics and identities, represents a major difficulty inthe direct study of the cellular biology of individual neurons or gliafrom this region of the brain. In particular, expression of specificneuropeptides, which characterise the identity of these unique neurons,are detected in relatively small populations of cells and, as evidencesuggests, are distributed throughout the region. Thus the study of themechanism of action of a specific neuropeptide, its gene regulation andboth its original or mediated roles and contributions within thehypothalamus is limited in situ. A way to attenuate this complexity andto investigate such questions at molecular and cellular level is theestablishment of clonal immortalized hypothalamic neuron- or glia-likecell lines that express the cellular markers of interest.

Historically it has proven to be difficult to establish immortalizedhypothalamic cell lines, due to the lack of naturally occurring CNStumors and the inherent difficulty of transforming or immortalizinghighly differentiated neurons from primary culture (2). Cell lines fromthe peripheral nervous system have been established from neuroblastomas,such as the Neuro2A cell line, and pheochromocytomas, such as the PC12cell line, however these models are not truly representative ofdifferentiated CNS neurons. Previous infection of primary cultures ofmouse hypothalamic tissue from embryonic day 14 with SV40 largeT-antigen in the early 1970's produced cell lines that were notconsidered fully differentiated (3). On the other hand, targetedtumorigenesis in transgenic mice has been used successfully to establishcell lines in specific tissues, such as the anterior pituitary andpancreas (4-9). In an attempt to produce a suitable model to study thegonadotropin-releasing hormone (GnRH) gene, a directed tumorigenesistechnique was used to develop a murine immortal cell line ofGnRH-secreting hypothalamic neurons (GT1 cells e.g. GT1-7 or Gn11) (10).The cells were developed by targeting expression of the potent oncogene,SV40 T-antigen, with the regulatory region of GnRH in transgenic mice(11). This cell line represents the only hypothalamic cell modelavailable for study and expresses a single neuropeptide, GnRH. Thus,there remains a real and unmet need for neuronal cell lines to providevalid model systems for molecular and biochemical investigations ofneuronal cells.

SUMMARY OF THE INVENTION

The present invention addresses the need for appropriate hypothalamiccell models for investigations at the cellular and molecular level.

It is an object of the invention to provide immortalized hypothalamicneuronal cell lines expressing specific markers characteristic ofindividual cell types and to a method for establishing same.

The present invention provides a series of clonal, immortalizedhypothalamic neuronal and glial cell lines that express a viraloncogene, and specific neuronal or glial markers. At least 36independent cell lines have been identified thus far which expressunique phenotypes.

In accordance with one aspect of the invention, there is provided amethod of making a hypothalamic cell line comprising:

-   -   (i) preparing a culture of embryonic hypothalamic cells;    -   (ii) infecting said culture with a retrovirus encoding a viral        oncogene, operably linked to a promoter and a selectable marker;    -   (iii) isolating transfected cells from non-transfected cells to        obtain a culture of immortalized hypothalamic cells;    -   (iv) subcloning said immortalized cells; and    -   (v) screening subcloned cells for expression of specific        neuronal markers.

In another aspect of the invention, there is provided a cell lineprepared according to the above-described method.

In one aspect, the embryonic cells are mammalian cells.

In a preferred embodiment, the embryonic cells are human.

In another preferred embodiment, the embryonic cells are murine cellsharvested at day 15, 17 or 18 of gestation.

In a further preferred embodiment, the viral oncogene is large T antigenof SV-40.

According to another aspect of the invention, there is provided a cellline of hypothalamic origin expressing a specific marker.

In a preferred embodiment, at least 90%, preferably 100%, of the cellsexpress the specific marker.

In another preferred embodiment, the cell line of the present inventionis used in transplantation.

The cell lines of the present invention are useful in the development ofanimal models of disease and in the treatment of disease.

In another aspect of the invention, there is provided an immortalizedcell line of murine hypothalmic neuronal cells comprising a geneencoding polyoma virus large T antigen operably linked to a promoter andexpressing at least one marker selected from the group consisting ofneuropeptide Y, gonadotropin-releasing hormone, growth-hormone releasinghormone (GHRH), TenM 1, 2, 3, 4, arginine vasopressin (AVP),thyrotropin-releasing hormone (TRH), SOCS-3, urocortin,melanocortin-concentrating hormone (MCH), orexin, dopamine transporter,corticotrophin-releasing factor (CRF), gonadotropin releasing hormonereceptor, tryptophan hydroxylase, tyrosine hydroxylase, galanin,proopiomelanocortin (POMC), proglucagon, neurotensin, somatostatin,agouti-related protein, cocaine and amphetamine-regulated transcript(CART), leptin, oxytocin, corticotrophin-releasing factor receptor 1 and2, aromatase, ghrelin, growth hormone secratogue receptor, androgenreceptor, estrogen receptor α, estrogen receptor β, leptin receptor,melanocortin-concentrating hormone receptor 3 and 4, neuropeptide Yreceptor Y1, neuropeptide Y receptor Y2, calcitonin receptor likereceptor, glucagon-like peptide 1 receptor, glucagon-like peptide 2receptor (Glp-2 receptor), and neurotensin receptor.

In one embodiment, the marker is Glp-2 receptor.

In another embodiment, the marker is neurotensin.

In another embodiment, the marker is proopiomelanocortin (POMC).

In another embodiment, the marker is neuropeptide Y (NPY).

In another embodiment, the marker is proglucagon.

In another embodiment, the marker is growth-hormone releasing hormone.

In another embodiment, the marker is urocortin.

In another embodiment, the marker is melanocortin-concentrating hormone.

In another embodiment, the marker is TenM 4.

In another embodiment, the marker is growth hormone secratogue receptor.

In another embodiment, the marker is ghrelin.

In a further aspect of the invention, the immortalized cell linecomprises a mixed cell population.

In another aspect of the invention, the immortalized cell line isprepared by the method comprising:

-   -   (i) preparing a culture of embryonic hypothalamic cells;    -   (ii) infecting said culture with a retrovirus encoding a viral        oncogene, operably linked to a promoter and a selectable marker;    -   (iii) isolating transfected cells from non-transfected cells to        obtain a culture of immortalized hypothalamic cells;    -   (iv) subcloning said immortalized cells into sub-cloned        populations;    -   (v) screening said subcloned populations for expression of        specific neuronal markers; and    -   (vi) selecting and further cloning a specific population.

In an embodiment of the invention, there is provided a method ofobtaining a neuropeptide comprising, culturing the cell line of theinvention that is known to express said neuropeptide and isolating theexpressed neuropeptide.

In one embodiment of the invention, there is provided a method foridentifying a modulator of a neuropeptide comprising:

-   -   (i) providing the cell line defined above;    -   (ii) incubating the cell line in the presence of the candidate        modulator; and    -   (iii) determining the biological effect of said candidate        modulator,        wherein said candidate is a modulator if it modulates the        neuropeptide expression and/or activity.

In another embodiment, the invention provides a method of identifying amodulator of a neuropeptide wherein said effect of said candidatemodulator can be determined by one of the following methods:

-   -   (a) monitoring effects on neuropeptide expression;    -   (b) incubating the said cell line with a substrate of a        neuropeptide and monitoring the effect on said substrate, such        as by monitoring metabolites of said substrate;    -   (c) binding assays; or    -   (d) proteomic profiling in the presence and absence of the said        candidate modulator.

In a further aspect of the invention, there is provided an immortalizedcell line of murine hypothalmic neuronal cells that is responsive to ateneurin C-terminal-associated peptide [TCAP].

In an embodiment of the invention, the teneurin C-terminal-associatedpeptide is selected from the group consisting of SEQ ID NOs 1-9.

In a further embodiment of the invention, the teneurinC-terminal-associated peptide is murine TCAP-1 or TCAP-3.

In another embodiment of the invention, the cell line is selected fromthe group consisting of N-7, N-22, N-29 and N-38.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described with respect to thedrawings, wherein:

FIG. 1 is a phase contrast microscopic view of the mixed neuronal cellpopulation, derived from day e17 fetal mouse hypothalamus, expressingSV40 large T-antigen (T-Ag);

FIG. 2 illustrates RT-PCR of the mixed cultures of immortalizedhypothalamic cultures (e15 and e17) derived using SV40 large T-antigenprimers (T-Ag, 433 bp fragment). Left panel: neuron-specific enolaseprimers (NSE, 391 bp fragment), and Right panel: neurofilament (NF).GT1-7 cell and hypothalmic RNA were used as positive controls, whereasreactions without reverse transcriptase (RT-) were also included.

FIG. 3A illustrates RT-PCR of cDNA from a number of subclonedhypothalamic cell lines initially using SV40 large T-antigen primers(T-Ag, 433 bp fragment);

FIG. 3B illustrates RT-PCR of the subcloned cell lines usingneuron-specific enolase primers (NSE, 391 bp fragment);

FIG. 4 illustrates cell line screening for CART, GHRH, AGRP, and leptinreceptor;

FIG. 5 illustrates cell line screening for NPY, galanin, proglucagon,POMC, and tyrosine hydroxylase;

FIG. 6 illustrates an agarose gel stained with ethidium bromide ofRT-PCR fragments for neurotensin, and mouse TenM neuropeptides 1, 2, and4 (New neuropeptide 1, 2 and 4);

FIG. 7 illustrates cell line screening for glp-2 receptor (G2R), andGnRH;

FIG. 8 illustrates cell line screening for CRF;

FIG. 9 demonstrates positive clones for GFAP;

FIG. 10 illustrates cell line screening for arginine vasopressin;

FIG. 11 illustrates the expression of ERα and ERβ;

FIG. 12 illustrates four phase contrast micrographs of NPY-17 neuronalcells (derived from day e17 fetal mouse hypothalamus) stained with 0.01%methylene blue;

FIG. 13 illustrates RT-PCR of NPY-17, GT1-7 and hypothalamic cDNA usingSV40 large T-antigen primers (T-Ag, 433 bp fragment), neuron-specificenolase primers (NSE, 391 bp fragment), neuropeptide Y primers (NPY, 282bp), and glial fibrillary acidic protein primers (GFAP, 621 bp);

FIG. 14 demonstrates specific staining with a mouse polyclonal NPYantibody (Peninsula Laboratories) in the NPY-17 neurons;

FIG. 15 illustrates the neurosecretory response of NPY-17 (N 38) cells.The effect of the addition of KCl (60 mM) on intracellular calciumlevels ([Ca2+]i) for the cells pictured is shown. Images of cells shownin the two panels on the right were obtained as per Example 5. Shown isa representative experiment from 6 experiments, n=116 cells in total(FIG. 15, left panel);

FIG. 16 illustrates the effect of sequential deletions of the NPY 5′regulatory region on expression in NPY-17 neurons;

FIG. 17 illustrates the expression of both estrogen receptor α (ERα) andestrogen receptor β (ERβ), and AR in the NPY-17 cells;

FIG. 18A illustrates the functional cAMP response of the immortalizedsubclones to TCAP peptide stimulation; and

FIG. 18B illustrates the response to glucagon-like peptide 2.

FIG. 19 illustrates that immortalized, clonal cell lines expressneuronal cell markers, but exhibit unique cellular morphologies andneuroendocrine markers. (A) Representative phase contrast micrographs ofclonal cell lines, N-1, N-4, N-6, N-20, N-36 and N-38. (B) Electronmicrographs of N-38 exhibiting dense core material (i, ii) and cellcontact regions (iii, iv). (C) RT-PCR of NSE, T-Ag, and GFAP, or (D)NPY, AgRP, and Ob-Rb in N-38 cells. GT1-7 cell and hypothalamic RNA areused as positive controls, whereas reactions without reversetranscriptase (RT-) were also included, as indicated.

FIG. 20 illustrates a comparison of human, murine and rainbow trout TCAPpeptide sequences [SEQ ID Nos 1-9].

DETAILED DESCRIPTION

The present invention provides a series of clonal, immortalizedhypothalamic neuronal cell lines that express the oncogene Simian virus(SV 40) large T-antigen and specific neuronal markers and a method forproducing same. A method for the generation of a number of clonal,immortalized hypothalamic neuronal and glial cell lines is alsoprovided. Several immortalized cells lines have been expanded andcharacterized.

“Immortalized cell line” as used herein means a cell line that canreplicate and be maintained indefinitely in in vitro cultures underconditions that promote growth, preferably at least over a period of ayear or years.

“Cell line” as used herein is a population or mixture of cells of commonorigin growing together after several passages in vitro. By growingtogether in the same medium and culture conditions, the cells of thecell line share the characteristics of generally similar growth rates,temperature, gas phase, nutritional and surface requirements. Thepresence of cells in the cell line expressing certain substances, forexample Neuropeptide Y (NPY) can be ascertained, provided a sufficientproportion, if not all, of cells in the line are present to produce ameasurable quantity of the substance. An enriched cell line is one inwhich cells having a certain trait e.g. expression of NPY, are presentin greater proportion after one or more subculture steps than theoriginal cell line. Preferably the cell line is derived from one, two orthree originating cells. The cell line can become more homogenous withsuccessive passages and selection for specific traits. Clonal cells arethose which are descended from a single cell. A cloned cell culture is acell culture derived from a single cell.

A SV40 Large T Antigen (SV-40 LTA) oncogene is intended to encompass anynucleotide sequence which encodes a protein having the function ofpolyoma (or SV-40) LTa and which is capable of being expressed in thehost cell in a quantity measurable by a known assay for LTa, such asimmunochemical staining.

A selectable marker is a genetic determinant which makes it possible toprovide culture conditions which favour the growth of cells possessingthe marker, compared to cells which do not. An antibiotic resistancegene is an example of a selectable marker.

The following abbreviations will have their standard scientificabbreviations: T-Ag, Large T-antigen; NSE, neuron-specific enolase;GFAP, glial fibrillary acidic protein; SNTX, syntaxin; ER, estrogenreceptor; AR, androgen receptor; LepR, leptin receptor b; Glp-2R (alsoG2R), glucagon-like peptide 2 receptor; SOCS-3, suppressor of cytokinesignaling 3; NPY, neuropeptide Y; AGRP, agoutirelated peptide; POMC,proopiomelanocortin; CART, cocaine and amphetamine regulated transcript;MCH, melanin-concentrating hormone; Ucn, urocortin; NT, neurotensin;Gal, galanin; Orx, orexin; DAT, dopamine transporter; CRFR,corticotrophin-releasing factor receptor; proGlu, proglucagon; GHRH,growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone;GnRHR, gonadotropin-releasing hormone receptor; CRF,corticotropin-releasing factor; TRH, thyroid-releasing hormone; AVP,arginine vasopressin; OXY, oxytocin; Arom, aromatase; TPH, tryptophanhydroxylase; TH, tyrosine hydroxylase; TenM-1 (also New-1); TenM-2 (alsoNew-2); TenM-3 (also New-3); and TenM-4 (also New-4), Teneurins 1-4;GHS-R, growth hormone secratogue receptor; Lep, leptin; SOM,somatostatin; NTR, neurotensin receptor; MC3R, melanocortin receptor-3;MC4R, melanocortin receptor-4; NPY-Y1, NPY receptor Y1; NPY-Y2, NPYreceptor Y2; CRLR, calcitonin receptor like receptor; nd, not done; na,not done; w, weak expression.

The present invention utilizes embryonic cells. For the purpose ofillustration herein, primary cell cultures from embryonic (e) day 18, 17and 15 fetal mouse hypothalamus were used representing a period ofrecognized neurogenesis. After preparing a single cell suspension, thecells are plated and are preferably allowed to grow for abouttwenty-four hours.

The primary cell cultures were then infected with a retrovirus whichharbours the sequence for SV-40 Large T antigen (T-Ag) and neomycinresistance. SV-40 (T-Ag) is known to cause tumor transformation. Apreferred virus according to the present invention is a replicationdefective, recombinant murine virus which is harvested from psitexcells.

The retrovirus infected cells are incubated in the presence of geneticin(G418) and resistant colonies are selected and expanded. After severalpassages, cells were subcloned and during each expansion step, a part ofthe culture was stored in liquid nitrogen.

A phase contrast microscopic view of the mixed neuronal cell population,derived from day e17 fetal mouse hypothalamus, expressing SV40 largeT-antigen is shown in FIG. 1. Cells were cultured on a cover slide in1×DMEM with 10% FBS (Gibco). Cells shown are at passage 3. Cells arestained with 0.01% methylene blue for 20 minutes, after fixation withparaformaldehyde. The magnification is 400×.

The mixed cell populations which comprise immortalized cells of morethan one phenotype are considered to be neuron-like due to theexpression of neuron-specific enolase (NSE), a specific neuronal marker,and neurofilament, but not glial fibrillary acidic protein (GFAP).

FIG. 2 illustrates the results of RT-PCR of the mixed cultures ofimmortalized hypothalamic cultures using SV40 large T-antigen primers(T-Ag, 433 bp fragment), neuron-specific enolase primers (NSE, 391 bpfragment), and neurofilament (NF). The control is RNA without thereverse transcriptase enzyme (RT-). PCR fragments were excised,subcloned, and sequenced to confirm identity. As a further control, theRNA used in all RT reactions was pre-treated with DNasel.

Further expansion of a cell line was performed only after the expressionof large T-Ag, neuron-specific enolase and lack of glial fibrillaryacidic protein was confirmed either through reverse transcriptasepolymerase chain reaction (RT-PCR) or Northern blotting.

Positive cell lines were further subcloned through successive dilutions.The clones were expanded and an aliquot was frozen in liquid nitrogen.RNA was extracted from each clone and used to make cDNA. The clonal cellline was then analyzed for expression of a large number of markersindicative of a unique neuronal phenotype. A correlation of cell linenames between those used in the figures and in Table 3 is listed inTable 1. A summary of the markers and cell lines screened are listed inTable 3. A list of the primers used for the screening of each marker islisted in Table 2.

The above described methodology generates a large library of potentialclones of interest. Preliminary experiments yielded approximately 36cell lines with a clonal (single) cell population or a cell populationof at most 2-3 cells, that were produced through serial dilutions of themixed populations of immortalized hypothalamic cells into 96 well tissueculture plates. Hypothalamic cells representing a mixed population werefrozen in liquid nitrogen. These immortalized hypothalamic cellpopulations represent a virtually unlimited resource of unique neuronalcell phenotypes and can be screened for any peptide, neurotransmitter,or receptor of interest. The present invention prevails a mixedpopulation of immortalized hypothalamic cells comprising a large T-Agand derived by the method of the present invention.

FIGS. 3A and 3B illustrate the results of RT-PCR of cDNA from a numberof subcloned hypothalamic cell lines initially using SV40 largeT-antigen primers (T-Ag, 433 bp fragment) (see FIG. 3A), andneuron-specific enolase primers (NSE, 391 bp fragment) (see FIG. 3B).

The cell lines were then screened for selected gene expression byRT-PCR, followed by Southern blotting in which, the blot was probed withan internal sequence to the flanking RT-PCR primers. Table 2 indicatesthe primers that were used. The control is RNA without the reversetranscriptase enzyme (RT-). PCR fragments were excised, subcloned, andsequenced to confirm identity. As a further control, the RNA used in allRT reactions was pre-treated with DNasel. Hypothalamic RNA and GT1-7GnRH neuronal RNA was used as a control in the screening.

FIGS. 4 to 11 illustrate the results of cell line screening for specificmarkers. FIG. 4 illustrates CART, GHRH, AGRP, and leptin receptorscreening. FIG. 5 illustrates NPY, galanin, proglucagon, POMC, andtyrosine hydroxylase screening. FIG. 6 is an agarose gel stained withethidium bromide of RT-PCR fragments for neurotensin, TenM neuropeptides1, 2, and 4. FIG. 7 illustrates screening for glp-2 receptor and GnRHand FIG. 8 illustrates the results of screening for CRF. Positive clonesfor GFAP are demonstrated in FIG. 9. FIG. 10 illustrates the results ofcell line screening for arginine vasopressin. FIG. 11 illustrates thepresence of PCR fragments corresponding to expression of both ERα andERβ.

Once it was determined that a cell line expresses a gene of interest,another 2 rounds of subcloning was done to be sure that the cellpopulation was indeed clonal and pure. These cells were then grown andfurther analysed to confirm the phenotype of the cell line. Eachindividual cell line was screened for an extensive list of markers.Table 2 provides a list of the neuropeptides and receptors for which thecell lines were screened. These markers are described below.

Cell lines that express these markers can be used as a source of saidpeptides. They can also be used in methods for identifying modulators ofsaid compound activity or substances that may be used in the treatmentof the peptide associated conditions. For instance, the cell culture ofthe cell lines can be incubated with a candidate modulator in order todetermine the effect on the respective marker expression and/or activityusing methodologies known in the art or discussed herein with respect toNPY cells and TCAP responsive cell lines.

Neuron-specific enolase (NSE) is the most abundant form of theglycolytic enzyme enolase found in adult neurons and is thought to serveas a growth factor in neurons. NSE is useful in studying neuronaldifferentiation and is, therefore, a valuable tool for visualizing theentire nervous and neuroendocrine systems. Serum levels of NSE have beenassociated with such disease states as Alzheimer's, Huntingdon Chorea,neuroblastoma, head trauma, neuroendocrine malignancies and small cellcarcinomas of the lung.

Glial fibrillary acidic protein (GFAP) is selectively located inastrocytes and represents the major constituent of astrocyticintermediate filaments. In adults, GFAP levels increase as a result ofthe proliferation of astrocytes that occurs in response to a variety ofphysical, chemical and etiological insults, including Alzheimer'sdisease, epilepsy and multiple sclerosis.

Estrogen receptors (ERα) are transcriptional factors with a DNA bindingdomain and ligand binding domain and have been shown to be expressed andtranscriptionally active in a number of tissues including ovary, testis,prostate and brain. The estrogen receptor has been classified into twodistinct isoforms, alpha and beta. ERα is known to interact in a liganddependent and independent manner with several known coactivatorsincluding Receptor-Interacting Protein 140 (RIP140/ERAP140), ERAP160,and Steroid Receptor Coactivator-1 (SRC-1) to enhance transcriptionalactivity of target genes.

Estrogen receptors (ERβ) are transcriptional factors with a DNA bindingdomain and ligand binding domain and have been shown to be expressed andtranscriptionally active in a number of tissues including ovary, testis,prostate and brain. ERβ has been shown to be a high affinity estrogenbinding protein capable of initiating transcription of genes under thecontrol of estrogen response elements (EREs).

Leptin receptor-b (LepR) is a class 1 cytokine receptor that isexpressed in high levels in the hypothalamus. Mutations in LepR havebeen found to cause obesity in mice and this receptor appears to becritical for the weight-reducing effects of leptin, a cytokine whichtargets various cells in the body.

Pro-opiomelanocortin (POMC) is a prohormone that acts as an importantmediator in the regulation of feeding behavior, insulin levels and,ultimately, body weight. Bioactive peptides derived from POMC aregenerated in neurons of the hypothalamus and act as endogenous ligandsfor the melanocortin-4 receptor (MC4R), a key molecule underlyingappetite control and energy homeostasis. It was determined that POMC isexpressed in cell lines N-4, N-11, N-19, N-20, N-22, N-29 and N-37, andweak expression was detected in cell lines N-3 and N-8 (see Table 3).These cell lines can be used in developing diagnostic and therapeutictreatments for disorders arising from dysregulation of feedingbehaviour, insulin levels, and body weight.

Galanin (Gal) is a neuropeptide that has been found to influence severalphysiological processes such as cognition and memory, the release ofvarious neurotransmitters and hormones (e.g. acetylcholine,noradrenaline, glutamate, dopamine, insulin, growth hormone, prolactin),motility of the digestive tract, nociception, feeding, and sexualbehavior.

Agouti-Related Protein (AGP) is a potent antagonist of themelanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) and isan integral component in the metabolic processes that regulate feedingbehavior and body weight.

Cocaine and amphetamine regulated transcript (CART) is a recentlydiscovered hypothalamic peptide with a potent appetite suppressingactivity. In the central nervous system CART is highly expressed in manyhypothalamic nuclei, some of which are involved in regulating feedingbehaviour.

Neuropeptide Y (NPY) is the most abundant neuropeptide in the brain. Itis a member of a family of proteins that include pancreatic polypeptide,peptide YY and seminalplasmin. In addition to its function in feedingbehavior, several other physiologic roles have been assigned to NPY,including involvement in circadian rhythms, sexual function, anxietyresponses and vascular resistance. It was determined that NPY isexpressed in cell lines N-29, N-38, N-25/2, N-29/4, N-40, N-41, N-42,N-43, N-44, N-45, N-46, and N-47, and weak expression was detected incell lines N-1, N-11, N-36 N-25/3 and N-29/1 (see Table 3). These celllines can be used in developing diagnostic and therapeutic treatmentsfor circadian rhythm, sexual function, anxiety and vascular resistancedisorders.

Growth hormone-releasing hormone (GHRH) is a mixture of two peptides,one containing 40 amino acids, the other 44. GHRH stimulates cells inthe anterior lobe of the pituitary to secrete growth hormone (GH). Itwas determined that GHRH is expressed in cell lines N-3, N-6, N-19,N-22, N-29, N-36, N-37 and N-38, and weak expression was detected incell lines N-1, N-2, N-8, N-11 and N-25 (see Table 3). These cell linescan be used in developing diagnostic and therapeutic treatments fordisorders including growth hormone-related disorders.

Corticotropin-releasing factor (CRF) has been hypothesized to beinvolved in the pathophysiology of anxiety, depression, cognitive andfeeding disorders. Two distinct CRF receptor subtypes, CRFR1 and CRFR2,are thought to mediate CRF actions in the central nervous system.

Urocortin (Ucn) is a peptide that binds and activates transfected type-1corticotropin-releasing factor (CRF) receptors and is more potent thanCRF at binding and activating type-2 CRF receptors. Indications are thaturocortin is an endogenous ligand for the type-2 CRF receptor, which ishypothesized to be involved in the pathophysiology of anxiety,depression, cognitive and feeding disorders. It was determined that Ucnis expressed in cell lines N-1, N-6, N-11, N-20, N-25, N-36, N-39,N-20/1, N-20/2, N-25/2, N25/3, N-29/1, N-29/2, N-29/3, N-29/4, N-36/1,N-36/2, N-40, N-41, N-42, N-43, N-46 and N-47, and weak expression wasdetected in cell lines N-3, N-, N-25/4, N-44, and N-45 (see Table 3).These cell lines can be used in developing diagnostic and therapeutictreatments for anxiety, depression, cognitive and feeding disorders.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide thatregulates a variety of functions in mammalian brain, in particularfeeding behavior. MCH is thought to influence feeding and energy balanceby acting downstream of leptin and the melanocortin system. It wasdetermined that MCH is expressed in cell lines N-3, N-4, N-7, N-29,N-38, N-39, N-20/1, N-20/2, N-25/2, N25/3, N-29/1, N-29/2, N-29/3,N-29/4, N-36/1, N-36/2, N-40, N-41, N-42, N-43, N-44, N-45, N-46 andN-47, and weak expression was detected in cell lines N-36, N-37 andN-25-4 (see Table 3). These cell lines can be used in developingdiagnostic and therapeutic treatments for feeding behaviour-relateddisorders.

The proglucagon gene (proGLU) encodes several hormones that areimportant in human physiology. Proglucagon-derived peptides are involvedin a wide variety of both peripheral as well as central functions, suchas glucose homeostasis, gastric emptying, insulin secretion and theregulation of food intake. It was determined that proGLU is expressed incell lines N-8, N-37, N-20/2, N-40, and N-41, and weak expression wasdetected in cell lines N-6, N-22, N-29 and N-39 (see Table 3). Thesecell lines can be used in developing diagnostic and therapeutictreatments for disorders relating to glucose homeostasis, gastricemptying, insulin secretion and the regulation of food intake.

Neurotensin (NT) is an endogenous peptide that is involved with memoryfunction, specifically Alzheimer's disease. This peptide may also beinvolved in the pathophysiology of Parkinson's disease andschizophrenia. It was determined that NT is expressed in cell linesN-36, N-39, N-20/1, N-20/2, N-25/2, N-29/1, N-29/2, N-29/3, N-29/4,N-36/1, N-36/2, N-40, N-41, N-42, N-43, N-44, N-45, N-46 and N-47 (seeTable 3). These cell lines can be used in developing diagnostic andtherapeutic treatments for Alzheimer's disease, Parkinson's disease andschizophrenia.

Oxytocin (OXT) is a neurohypophyseal hormone that has a wide range ofbehavioral effects outside its classic peripheral endocrine functions.OXT involvement in adaptive central nervous system processes has beendemonstrated as an inhibitory, amnestic action on learning and memory indifferent paradigms.

Arginine vasopressin hormone (AVP) (also known as anti-diuretic hormone,ADH) is important in the regulation of the water permeability of renalcollecting tubules and the ascending loop of Henle. It is also avasoconstrictor and is thought to play a role in arterial pressuremaintenance during blood loss.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in thebiosynthesis of serotonin and an important component of melatoninbiosynthesis. Serotonin functions mainly as a neurotransmitter, whereasmelatonin is the principal hormone secreted by the pineal gland. The TPHgene has recently been associated with behavioural disorders such asmanic depression and aggression.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in thebiosynthesis of catecholamines, an example being dopamine. The enzymehas been intensively studied in relation to both its physiologicalfunction in the brain and brain disorders. The study of this enzyme hasled to the understanding of many diseases such as Parkinson's disease,stress and emotional disorders.

SOCS-3 is an inhibitor of cytokine receptor signaling that blockssignals from the brain that normally shut down appetite when the body'sfat cells have had their fill.

Androgen receptors (AR) have been found in a variety of tissues,including reproductive organs, central nervous system and skeletalmuscle.

Orexins (Orx) are a family of hypothalamic neuropeptides selectivelyexpressed in the hypothalamus that are thought to play a role in feedingbehaviour.

The dopamine transporter (DAT) is the site of presynaptic reuptake ofdopamine, an event that terminates its synaptic activity.

Corticotrophin-releasing factor receptor-1 (CFFR1) is thought to mediateCRF actions in the CNS. Genetic deletion of the CRFR1 receptor can leadto impairments in anxiety-like and cognitive behaviors, supporting acritical role for this receptor in anxiety and cognitive biologicalprocesses.

Corticotrophin-releasing factor receptor 2 (CRFR2) is thought to mediateCRF actions in the CNS.

Aromatase is responsible for converting testosterone into estrogens.

Gonadotropin releasing hormone receptor (GnRH Receptor) is the receptorfor gonadotropin releasing hormone, a neuropeptide that is involved inregulating reproduction.

Gonadotropin releasing hormone (GnRH) is a neuropeptide that is involvedin regulating reproduction. The release of GnRH from the hypothalamusregulates the production of gonadotropins in the pituitary and thesegonadotropins are responsible for gonadal development and growth invertebrates.

Leptin is a protein hormone with important effects in regulating bodyweight, metabolism and reproductive function. The protein isapproximately ˜16 kDa in mass and encoded by the obese (ob) gene.

Ghrelin is an endogenous ligand for the growth hormone seretagoguereceptor (GHS-Rs), which regulates pituitary growth hormone (GH)secretion. It is associated with obesity and has shown to be involved inthe regulation of food intake and body weight. It was determined thatghrelin is expressed in cell lines N-1, N-7, N-8, N-22, N-37, N-38,N-39, N-20/1, N-20/2, N-25/2, N-29/1, N-29/2, N-29/3, N-29/4, N-36/1,N-36/2, N-40, N-41, N-42, N-43, N-44, N-45, N-46 and N-47 (see Table 3).These cell lines can be used in developing diagnostic and therapeutictreatments for obesity and disorders involving the regulation of foodintake and body weight.

Growth hormone secratogue receptor (GHS-R) is a G-protein-coupledreceptor that is expressed in the pituitary gland and in several areasof the brain including the hypothalamus. It is also known as the Ghrelinreceptor. It was determined that GHS-R is expressed in cell lines N-39,N-29/1, N-29/2, N-29/3, N-44 and N-46 (see Table 3). These cell linescan be used in developing diagnostic and therapeutic treatments.

Neurofilament protein (NF) is a marker of neurons, mainly axons.

Somatostatin (SOM) is a hormone that inhibits the secretion of growthhormone. Somatostatin and its synthetic analogs are used clinically totreat a variety of neoplasms. It is also used in to treat giantism andacromegaly, due to its ability to inhibit growth hormone secretion.

Estrogen receptor-associated protein 140 (ERAP-140) is a protein thathas been found to interact with the estrogen receptor in a hormonedependant manner.

TAU represents a family of microtubule-associated proteins that arethought to regulate different properties of neuronal (and possibly alsoglial) microtubules, especially their stability and orientation. Becausethe spreading of intraneuronal deposits of an altered form of thisprotein correlates with the severity of Alzheimer symptoms, it isthought to be implicated in the pathogenesis of Alzheimer's disease.

Neurotensin receptor (NTR) is a G-protein coupled receptor forneurotensin, which is an endogenous neurotransmitter that influencesdistinct central and peripheral physiological functions in mammals.

Melanocortin receptor-3 (MC3R) is a receptor for melanocortin hormone,which is involved in the regulation of satiety and energy homeostasis.

Melanocortin receptor-4 (MC4R) is a receptor for melanocortin hormone,which is involved in the regulation of satiety and energy homeostasis.

NPY Receptor Y1 (NPY-Y1) is a receptor for NPY that is involved inmediating stimulatory effects on food intake.

NPY Receptor Y2 (NPY-Y2) is a receptor for NPY that is involved inmediating stimulatory effects on food intake.

Calcitonin Receptor Like Receptor (CRLR) is a receptor forCalcitonin-gene-related peptide (CGRP), a peptide with many actionsranging from vasodilation to inhibition of some of the effects ofinsulin on metabolism. It has 55% homology with the calcitonin receptor.

Glucagon-like peptide 2 receptor (Glp-2R or G2R) is a member of asubfamily of G protein-coupled receptors. Glp-2R is expressed on neuralcells and its expression has been found in the cerebellum, medulla,amygdala, hippocampus, dentate gyrus, pons, cerebral cortex, pituitarygland, and hypothalamus. Glp-2R is thought to have a role in tasteaversion and nausea, and its signaling has been shown to enhance cellsurvival and inhibit apoptosis. It was determined that Glp-2R isexpressed in cell lines N-7, N-19, N-22, and N-29 and weakly expressedin cell line N-3 (see Table 3). These cell lines can be used indeveloping diagnostic and therapeutic treatments.

The teneurins are the vertebrate homologues of an enigmatic family ofproteins (Ten-m) originally discovered in Drosophila (33-35). Thehighest levels of the ten-m genes occur in the central nervous systemwhere the protein occurs preferentially on the surface of axons (35,36).Mutations of the Drosophila ten-m gene result in embryonic lethality(34,35). Four such teneurin paralogous genes exist in vertebrates andencode approximately a 2800-amino acid Type II transmembrane proteinwhere the carboxy terminus of the protein is displayed on theextracellular face of the cell (32). Teneurin-4 in mammalian cells isupregulated by the transcription factor GADD153/CHOP (37), atranscription factor that is induced by several types of cellular stressor conditions triggering endoplasmic reticulum (ER) stress.Overexpression of teneurin-2 into the mouse neuroblastoma cells (Nb2a)can increase the amount of neurite outgrowth and enlarge the growthcones. Family members include TenM 1, TenM 2, TenM 3 and TenM 4. It wasdetermined that TenM 4 (New-4) is expressed in cell lines N-1, N-2, N-3,N-4, N-6, N-8, N-11, N-19, N-29, N-36, and N-37 (see Table 3). Thesecell lines can be used in developing diagnostic and therapeutictreatments.

The screening generally included: Southern blotting with an internalprobe to the gene of interest; cloning and sequencing of the PCR productto ensure that the DNA fragment is truly the gene of interest; andNorthern analysis to determine the expression levels of the gene ofinterest. Functional studies, depending upon the gene expressionprofile, were also performed to assess the status of each cell line.

As summarized in Tables 1-3, a number of individual cell lines withexpression of a number of neuropeptides, enzymatic markers, and receptormolecules have been found. RT-PCR results, followed by Southern blot andsequencing confirmation, indicates that at least one cell lineexpressing each of: neuropeptide Y, gonadotropin-releasing hormone,tyrosine hydroxylase, galanin, proopiomelanocortin (POMC), proglucagon,neurotensin, somatostatin, urocortin, growth hormone-releasing hormone,melanocortin-concentrating hormone and agouti-related protein has beencharacterized. It is clearly apparent that the initial library of mixedpopulation of immortalized cells contains many other potential cloneswhich have not yet been analyzed due to the sheer volume generated bythe method. However, the mixed population of immortalized cells isuseful for studying the interneuronal communication of cells present inthe hypothalamus and for screening the effects of various substances andconditions on said mixed population of cells. These cell lines can beused in the methods described herein for the more specific cell lines onmicroarray analysis and through proteomic profiling and in developingdiagnostic and therapeutic treatments for various neuropathologicaldisorders. Several of the neuropeptide-expressing cell lines which wereanalysed, did have receptors for androgen and estrogen, leptin,melanocortin-concentrating hormone receptor 3 and 4, neuropeptide Y.glucagon-like peptide 1, glucagon-like peptide 2, and neurotensin.

NPY-17 (N-38) Cell Line

One particular subcloned cell line, NPY-17, derived at e17, synthesizesNPY and expresses neuronal, but not glial, cell markers and thereceptors for a number of neurotransmitters and neuromodulators thoughtto be involved in the control of the NPY neuron. Neuropeptide Y isabundantly expressed in the central nervous system and is highlyconserved across species (13). It has a broad range of physiologicalactivities. The most recognized functions include the regulation ofendocrine function, circadian rhythms, and satiety, but it has also beenimplicated in psychiatric disorders, stress response and as acardiovascular regulator or vasoconstrictor (13). Central administrationof NPY stimulates feeding and repeated doses results in an increase inbody weight (14). Hypothalamic NPY and GnRH neurons display many commoncharacteristics, as do other neurosecretory cells. The NPY gene encodesa prepro-NPY precursor, NPY is stored in secretory granules, and it issecreted in a pulsatile manner, at a similar frequency to GnRH (15, 16).The NPY gene 5′ regulatory region has been partially defined usingtransient transfection analysis in PC12 cells, a pheochromocytoma cellline that can differentiate into neurons -upon treatment with nervegrowth factor (NGF), or LA-N-5 cells, a neuroblastoma cell line (17-20).The PC12 cell line is certainly not representative of a hypothalamic NPYneuron as the tumor originated in the adrenal gland and only expressesNPY after the cells are treated with NGF to promote neuronaldifferentiation. Nevertheless much of the research regarding NPY geneexpression has been performed with this cell line. It is known frombilateral neural transection experiments and antisense data that the NPYneurons responsible for the reproductive and orexigenic effects of NPYlie within defined regions of the hypothalamus (21-23), which clearlyare not represented by any of the tumor-derived cell lines used for theNPY studies. Further, since differential NPY gene expression has beenproposed to be region and cell specific, we are poised to produce whatwould appear to be more representative data with the NPY-17 hypothalamiccell line. Interestingly, regulation of the gene by forskolin, a PKAactivator, and phorbol ester, a PKC activator, has been found to besimilar but through unique mechanisms in the two cell lines used for NPYstudies, PC12 and LA-N-5 cells (24), indicating that there is cellspecific regulation of the NPY gene.

This is the first report of the generation of a clonal, hypothalamic,immortalized NPY-producing neuronal cell line. The immortalizationprotocol of the present invention produced a number of different cellmodels of clonal hypothalamic neurons and glial cells. AnNPY-synthesizing clone (NPY-17) was first selected for furthercharacterization, due to the important role that NPY plays in thecontrol of reproductive physiology.

In spite of the fact that NPY itself has such an extensive repertoire ofphysiological functions, relatively little research has been done withregards to the molecular mechanisms involved in the regulation of NPYtranscription. Further, as previously mentioned, much of the work thathas been done involved heterologous or tumor cell lines, withtransfection of the NPY promoter sequences, or in brain slices/primaryculture, which contain a mixed population of neurons. This is due to thefact that no suitable model of the hypothalamic NPY neurons wasavailable for these studies. With the development of the NPY-17 cellline, it is possible to approach these studies in a clonal population ofNPY-expressing cells.

The NPY-17 cells are stable in culture, with a doubling time of 1-2days. They can be passaged extensively, maintaining expression of SV40T-antigen. Morphologically, they appear to possess a neuronal phenotype.They have clearly defined perikarya and neurites. Some of the neuritesappear as short dendritic-like processes, while others form lengthyprocesses. The cells are fairly large and globular, and appear to have alarge nucleus, when compared to the GT1-7 neuron which are moreelongated.

FIG. 12 is a phase contrast micrograph of NPY-17 neuronal cells (derivedfrom day e17 fetal mouse hypothalamus). Cells were cultured on a coverslide in 1×DMEM with 10% FBS (Gibco). Cells shown are at passage 5 afterpurification. NPY-17 cells are stained with 0.01% methylene blue for 20minutes, after fixation with paraformaldehyde. The magnification is400×.

Preliminary characterization of this cell line, NPY-17, indicates thatit expresses only neuronal cell markers, such as neuron-specific enolaseand the 68 kDa neurofilament protein, but not glial fibrillary acidicprotein, normally found in astrocytes.

FIG. 13 illustrates RT-PCR of NPY-17, GT1-7 and hypothalamic cDNA usingSV40 large T-antigen primers (T-Ag, 433 bp fragment), neuron-specificenolase primers (NSE, 391 bp fragment), neuropeptide Y primers (NPY, 282bp), and glial fibrillary acidic protein primers (GFAP, 621 bp). Controlis NPY-17 cDNA without the reverse transcriptase enzyme (RT-). PCRfragments were excised, subcloned, and sequenced to confirm identity. Asa further control, the RNA used in all RT reactions was pre-treated withDNasel.

The NPY-17 cells express NPY, as detected by RT-PCR andimmunocytochemistry. As shown in FIG. 14, immunocytochemistry with amouse polyclonal NPY antibody (Peninsula Laboratories) displays specificstaining in the NPY-17 neurons (i). NPY-17 cells were plated onpoly-L-lysine coated coverslips, washed with PBST, and preincubated withgoat serum. Cells were then incubated with the NPY primary (1°) antibody(Peninsula Labs) for 24 hours (iii), followed by secondary antibody.Immunoreactivity was visualized with a Texas Red fluorescent tag. Cellnuclei are visualized with DAPI fluorescence (ii). Parallel staining wasdone with no incubation with 1° NPY antibody (iv and v).

Using RT-PCR, it was found that the NPY neurons express estrogenreceptors, α and β, and androgen receptors. FIG. 17A illustrates RT-PCRof NPY-17 and GT1-7 cDNA using ERα primers (344 bp fragment) andinternal ERβ primers (332 bp fragment). The ERβ fragment was too weak tovisualize with the ERβ external primers, so the external ERβ PCR productwas reamplified with internal primers to ERβ. The control is NPY-17 cDNAwithout the reverse transcriptase enzyme (RT-). PCR fragments wereexcised, subcloned, and sequenced to confirm identity. FIG. 17Billustrates RT-PCR of NPY-17 and GT1-7 cDNA using AR primers (560 bpfragment). The control is NPY-17 cDNA without the reverse transcriptaseenzyme (RT-). As a further control, the RNA used in all RT reactions waspre-treated with DNasel.

The NPY-17 cells also express leptin receptors and have been studied forleptin responsiveness using both gene array analysis and proteomicanalysis. NPY-17 cells were treated with 10-7 M leptin (recombinantmouse, Bachem Scientific) for 20 hours. Cells were washed with PBS andharvested through centrifugation. Nuclear extracts were prepared andexposed to mass spectrometry analysis. Preliminary analysis of the dataindicates that approximately 250 proteins are altered by leptintreatment. The analysis is not considered quantitative, but initiallyrepresents a +/− protein status (i.e. the protein is either present orabsent in the control or leptin-treated sample).

Since many of the peptides and enzyme present in the individual celllines are secretory products, one representative cell line, N-38 (Alsoknown as NPY-17), was studied for its response to depolarization byaddition of 60 mM KCl. FIG. 15 illustrates that the NPY-17 cells exhibitan intracellular calcium response to KCl, indicating that they areneurosecretory cells. NPY-17 neurons were plated in 60 mm plates.Fura-2/AM loaded GT1-7 neurons were continuously monitored by 340/380ratiometric digital imaging pre (top right panel) and post (lower rightpanel) stimulation. A representative graph is provided (FIG. 15, leftpanel) showing the effect of KCl (60 mM) on ([Ca2+]i) as a function ofraw 340/380 nM ratios for n=12 cells. KCl (60 mM) caused a significantincrease in calcium mobilization in the NPY-17 cells. The representativegraph of [Ca2+]i shows a peak value of ˜450 nM. The single spikeobserved is typical of a functional neurosecretory response.

It has also been found that sequential deletions of the NPY 5′regulatory region affect differential expression in NPY-17 neurons.NPY-17 cells were transiently transfected with 15 μg of one of theexpression vectors containing the human NPY 5′ regulatory region (−1078bp) and sequential deletion mutants of this same region. Transfectionswere incubated for 48 hours after the primary transfection of the DNAfor 12 hours with the calcium phosphate precipitate. Bars represent therelative LUC activity as compared to vector control activity (pGL2-luc)of each construct. The preliminary data is an average of two independentmeasurements in duplicate or triplicate.

Such cells can be used to identify modulators of NPY. Such modulatorscan then subsequently be used to treat NPY-related conditions.

TCAP Responsive Cell Lines

The onset of mood disorders, such as depression or anxiety, involve thealtered function of multiple loci in the brain that regulateemotionality, memory and motivation (41-43). However, the inability todescribe a cellular and molecular aetiology of these conditions likelyreflects, in part, the existence of critical neurological circuits notyet understood. A novel family of neuropeptides on the carboxy-terminusof the teneurin transmembrane proteins (32, 44) is implicated inneuronal communication. In one embodiment, they modulate stressresponses and anxiety (data not shown) and likely play a role in somepsychiatric illnesses. In another embodiment, they are implicated asmodulators of cell cycle and cell proliferation. These sequences arecalled teneurin C-terminal-associated peptides (TCAP-1 to 4) [SEQ. IDNOs 1-9]. These peptides may also have a cleavable amidation signal atthe C-terminus, such as “GRR” or “GKR”. The immortalized cell lines werescreened for TCAP responsiveness using rtTCAP-3 [SEQ. ID NO 9] plusamidation signal GKR. TCAP signals through a specific cAMP-dependentG-protein-coupled receptor to modify cell cycle and proliferation inimmortalized neurons by regulating genes associated with growthsuppression and promotion, and neuronal signaling.

The functional cAMP response of the immortalized subclones to TCAPpeptide stimulation was also assessed and the results are shown in FIG.18. N-7 (N-15-1, #7), N-22 (N-18-1, #11), N-38 and N-29 (N-15-14, #29)have been identified as TCAP responsive cell lines. N-7, N-22 and N-29were analyzed for the cAMP response to peptide stimulation. Thesubclones were split into 24 well plates. Cells were starved for 1 hourin DMEM without FBS, then medium was replaced with 0.5 ml fresh DMEM(without FBS) with the compounds as indicated. In FIG. 18A, neurons wereexposed to 10⁻⁷ M (100 nM) TCAP peptide (a newly discoveredstress-regulating peptide provided by Dr. D. Lovejoy at the Universityof Toronto), rtTCAP-3 [SEQ ID NO 9]. In FIG. 18B, they were exposed toglucagon-like peptide 2 at 0.2, 2, or 20 nM for 15 minutes. All peptideswere diluted in DMEM containing IBMX (100 μM). After a 15 minuteincubation at 37° C., 1 ml of ice-cold ethanol was added to each well.Cells were scraped from the plate and kept at −20° C. until the amountsof intracellular cAMP were determined in triplicate by RIA(Biotechnologies Inc., Stoughton, Mass.) according to the manufacturer'sinstructions.

Further, to demonstrate that the cell lines can be used as a model forstudying TCAP responsiveness, modulation, and in screening for TCAPmodulators, microarray studies were performed on 1 nM TCAP-1 [SEQ ID NO5 plus amidation signal GRR at C-terminus] treated N38 hypothalmiccells, which do not possess either CRF receptor subtype (Table 4). RNAsisolated from treated and untreated cells were analyzed onoligonucleotide arrays representing 12,884 mouse genes (Affymetrix,http://www.affymetrix.com). Standard filtering (p<0.005) andhierarchical clustering algorithm (average linage method: GeneSpringsoftware—Silicon Genetics) identified significant changes in theexpression of 4, 841/12,885 genes with 166 genes showing 1.5 folddown-regulation and 35 genes up-regulation in the TCAP-1-treated cellscompared to the untreated cells. At 16 hours post-treatment, asignificant decrease occurred among several genes, notably, GAS5, SDPRand CD95 that have been associated with growth arrest or apoptoticevents (45-47). In contrast, upregulated genes including MK167, MOP3 andGDAP10 have been associated with cell proliferation and cell cyclemodulation (48-50). A G-protein coupled receptor-related signaltransduction pathway is indicated by the regulation of genes, CREM,AKAP8, AKAP95 and PDE6A. Downstream effectors of RAS such as EFK1 andRGL were also down regulated. Downregulation of the A kinase anchoringprotein AKAP95 but upregulation of AKAP8 suggests that TCAP may act, inpart, by changing the targeting pattern of PKA (51). The upregulation ininducible nitric oxide (INOS), a intracellular voltage-gated chloridechannel (CLCN3) and the serotonin transporter (SLC6A4) may reflect thedown stream actions of cAMP-mediated signal cascade and indicates thepotential for TCAP to be involved in neuronal signaling systems. A rolein interneuron communication by TCAP is also indicated by the modulationof genes associated with the regulation of vesicle trafficking. Thus,the TCAP responsive cell lines can be used to screen for modulators ofneuronal function that affect growth, differentiation and communication.

While certain cell lines, such as NPY-17, and TCAP responsive cell linesN-7, N-22, N-38 and N-29, have been particularly described, the presentinvention is not limited to only these cell lines. The invention alsoprovides the cell lines listed and characterized in Table 3.

Applications

Using the immortalization, selection and screening methods of thepresent invention, many unique immortalized hypothalamic cells, of bothneuroendocrine and glial origin, have been isolated, a few of which areindicated in Table 3. The cells generated by the method of the presentinvention are unique compared to other immortalized cell lines becausethe cells did not originate as a tumor, but were transformed only by theexpression of SV40 large T-antigen in monolayer primary culture. Thetransformation process therefore is kept to a minimum (it has beenpostulated that tumorigenesis often results from more than one geneticevent), which should be more representative of the primary cell. Assuch, the present invention provides a method for generatingimmortalized hypothalamic neuronal cell lines, more preferably derivedfrom embryonic cells. In another embodiment the cell lines are derivedfrom murine cells. Further the cells are considered to be neuron-likedue to the expression of neuron-specific enolase, a specific neuronalmarker (3). These immortalized, clonal cell lines provide valid modelsystems for molecular and biochemical investigations on the regulationof specific hormones, characteristics of their respective secretoryneuronal population and an unlimited source of homogenous cell materialand of the neuropeptide itself. The method of the present invention hasbeen used to isolate a virtually unlimited number of cell models fromthe hypothalamus by immortalizing hypothalamic primary cell cultures. Anumber of the immortalized cells have been cloned and characterized.

For instance, the cell lines of the present invention can be used toidentify modulators of respective neuropeptides, that may for instancebe used in the treatment or regulation of a disorder associated with thesaid neuropeptide. This can be done by incubating the cells of the cellline with the candidate modulator under conditions that promoteneuropeptide expression and then monitoring the effect of the candidatemodulator on the neuropeptide. Suitable monitoring techniques are knownin the art, such as monitoring neuropeptide expression (e.g. mRNA orpeptide level), and/or neuropeptide activity (38, 39) Neuropeptideactivity can be monitored by incubating the cell line under conditionsthat promote neuropeptide expression, in the presence of a knownneuropeptide substrate and the candidate modulator and monitoring theeffect of the candidate modulator on neuropeptide activity and/orexpression. Such effect can be compared to a control level (internal orexternal controls). For instance the control can be neuropeptideactivity/expression in the absence of the candidate modulator, in thepresence of a known modulator, and/or in just buffer, etc. The presentinvention is not limited to such controls. Alternatively, such assayscan be performed under conditions that inhibit neuropeptide expressionand the effect of the candidate modulator can be assessed as above.Modulation is observed when there is an increase, decrease orhomeostasis effect on the neuropeptide expression and/or activity.

The cell lines and related methods and modulator screening assays of thepresent invention, for instance as noted above, are useful for manyapplications. A few areas where these cell lines are useful include drugdiscovery, therapeutic drug testing platforms, phenotypic profiling ofindividual neuronal cell types, gene expression studies, studies ofmechanisms of action of novel neuropeptides and neuronal mechanisms,signal transduction in neurons, and receptor cloning andcharacterization, neuronal regeneration and cell death. They are alsouseful in proteomic studies, genome-wide gene expression profiling inselected neuronal cell types, neuron-specific transcription factoranalysis, neuron-neuron interactions and communication, brain modellingand studies of ion channel function. The cell lines are also useful forstudies in neuronal regeneration and cell death, as well as in animalmodels of disease. The cell lines can also be useful in identifyingdiagnostic tests for certain medical conditions. As each immortalizedcell line of the present invention corresponds to an actual neuronalcell type, these endogenous cell types can be isolated using endogenouscharacteristic markers identified on the respective immortalized cellline (38-40). Specific cell lines of the present invention or theendogenous non-immortalized cell types identified and isolated may alsobe useful in the treatment of certain disorders or diseases.

The cell lines can also be used as a source of neuropeptides which inturn can be used in medical treatment, and in screening for modulatorsof said peptide and in drug design. One aspect of drug design would bebased on structural and chemical characteristics of the neuropeptide.For instance, a number of the neuronal cell models reported herein havethe potential to produce major neurotransmitters, as many of the linesexpressed tyrosine hydroxylase, a marker of catecholaminergic neurons.Cells expressing tyrosine hydroxylase have the potential to producedopamine, norepinephrine, and epinephrine, depending upon the complementof downstream catalytic enzymes. Other cell lines expressed tryptophanhydroxylase, the rate-limiting enzyme of serotonin production and animportant component of melatonin biosynthesis. Due to the involvement ofthese neurotransmitters in the development of neurological disorders,such as depression, the use of the cell lines to study their regulationis fundamental for the development of satisfactory treatment anddiagnostic options. Cell lines were also generated expressing specificreleasing peptides, such as growth hormone-releasing hormone, GnRH, orsecreted peptides, such as proglucagon-derived peptides, oxytocin orarginine vasopressin.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Methods of tissue culture, protein and peptide biochemistry, molecularbiology, histology and immunology referred to but not explicitlydescribed in this disclosure and examples are reported in the scientificliterature and are well known to those skilled in the art.

Example 1 Primary Cell Culture of e15, 17, and 18 Hypothalamus

Mice (BALB/c females and DC1 males from Charles River) were bred and theappearance of a vaginal plug signified embryonic day 0. An entire litterwas harvested at embryonic day 15, 17 or 18. Gravid mice were euthanizedby CO₂ inhalation. The uterus was removed immediately and placed in a100 mm tissue culture dish containing phosphate buffered saline (PBS),with 1 mM EDTA, pH 7.4, at 4° C. The dish was transferred to adissecting microscope where the fetuses were removed from the uterus.The fetuses were decapitated and the heads transferred to a new culturedish containing primary culture medium (DMEM, 10% heat-inactivateddefined FBS, 10% heat-inactivated defined horse serum, 1%penicillin-streptomycin, and 20 mM D-glucose (all from LifeTechnologies/Gibco)). Osmolarity of the culture medium was adjusted to320-325 mOsm with glucose. The plate contained enough culture medium tocover the head, and no more than three heads per dish were maintained.Dissection was optimized for speed and accuracy. Two needle-pointforceps were used to separate meninges and the hypothalamus wastransferred to a sterile 15 ml tube containing 1 ml of culture medium at4° C. Tissue was dissociated into a single cell suspension bytrituration with 2-3 fire-polished Pasteur pipettes of decreasing tipdiameter (4-6 strokes per pipette). The cell suspension was split intotwo 75-80 cm² tissue culture flasks, coated with 100 μg/ml poly-L-lysine(Sigma P-6282), with 8 ml of culture medium. Cells were allowed toattach to the flask for 24 h incubating at 37° C. with 5% CO₂. After 24h of growth the cells were ready for infection with retrovirus.

Example 2 Retroviral Infection of Hypothalamic Primary Cell Cultures

Because extreme caution should be used when handling retroviral culturesharbouring oncogenic immortalization factors, such as simian virus largeT-antigen, a Level 3 containment room and tissue culture hood was usedfor this work. The viral supernatant was prepared at a titre between10⁵-10⁶ cfu/ml. The producer cells harbouring the oncogene (describedbelow) were co-cultured with NIH3T3 cells (host) at a 1:4 ratio. Theviral supernatant was collected and assayed for colony forming units/mlof supernatant. The supernatant was stored at −80° C. until primary cellculture infection. After attachment of primary cells to the culture dishfor approximately 24 h, cells were incubated for 1 h, twicesuccessively, with fresh virus-containing medium harvested fromconfluent culture of ψ2 cells (psitex cells) producing areplication-defective, recombinant murine retrovirus. This virus,constructed by using the pZIPNeo SV(X) 1 vector, harbours the intactcDNA sequence for simian virus (SV40) large T antigen and neomycinresistance gene. Retrovirus infected cells (after 48 h in culture mediumwith retrovirus) were incubated with medium containing geneticin (G 418)in highly selective concentration (400-600 μg/ml for initial selection,250 μg/ml for cell maintenance). Geneticin selection of infected cellswas performed parallel with geneticin treatment of non-infected cells asa control.

Example 3 Analysis of Cell Populations and Subcloning of Cell Lines

Resistant colonies, appearing after 2-3 weeks, were picked using cloningcylinders and further expanded (40). After several passages cells weresub-cloned and during each expansion step, a part of the culture wasfrozen in cryoprotectant medium and stored in liquid nitrogen. Thegrowth curves of cloned cell lines, still representing a mixedpopulation of hypothalamic cells, displayed a doubling time ofapproximately 24-48 h. The new clones were selected if they demonstrateda predominantly neuronal lineage morphology, as small, rounded or ovoidperikarya and long neuritic processes. Generally, the cloned lines formmonolayers, grow rapidly and retain growth contact inhibition. Furtherexpansion of a cell line was performed only after the evidence for theexpression of at least large T antigens neuron-specific enolase and lackof glial fibrillary acidic protein, determined either through reversetranscriptase polymerase chain reaction (RT-PCR) reaction or Northernblotting. A cell line with a mixed population of hypothalamic cells wasfurther subcloned through successive dilutions of the trypsinized cellsinto 96 well tissue culture plates coated with poly-L-lysine. Theoptimal dilution allowed only 1 or 2 cells per well. The cells wereincubated in conditioned medium, i.e. medium taken from the mixedcultures, at a 1:1 ratio with culture medium, DMEM with 15% FBS (LifeTechnologies/Gibco). Cell colonies were allowed to grow and thensuccessively split into 24 well plates, and finally 60 mm plates. Theseclones were grown until the cells could be frozen in liquid nitrogen andalso harvested for RNA extraction. The RNA was used to make cDNA, andthe clonal cell line was analysed for expression of a large number ofmarkers indicating a unique neuronal phenotype. Immortalized cell lineswere grown in DMEM supplemented with 10% FBS, 20 mM glucose andpenicillin-streptomycin maintained at 37° C. with 5% CO₂.

Example 4 Cell Line Screening

Each cell line was analyzed for the expression of specific markers byreverse transcriptase PCR (RT-PCR). First strand cDNA was synthesizedfrom 10 μg of DNasel-treated RNA, using SuperScript II reversetranscriptase (RT) (Life Technologies/Gibco). The RT reaction was primedwith random primers. CDNA synthesis was followed by RNase H (180 U/ml)digestion of RNA in a total volume of 20 μL. Control reactions wereperformed where amplification was carried out on samples in which the RTwas omitted (RT-). Whenever possible, primer sequences flanked anintron, as an extra control for DNA contamination. In most cases, theagarose gels were transferred for Southern blot analysis with aninternal primer sequence as a probe. All products were sequenced toconfirm identity. Amplification details can be found in Table 2.

Example 5 Calcium Imaging

Ca2+ imaging experiments were performed with Fura2 (Molecular Probes)using an Olympus IX70 inverted epifluorescence microscope, an Ultrapixcamera with an EEV CCD37-10-1-019 chip, a monochromator, and a PCcomputer with Merlin imaging software (Life Science Resources) usingstandard Fura2 optics and imaging techniques. The extracellular solutionused for imaging experiments consisted of (in mM): 140 NaCl, 4 KCl, 2CaCl₂, 1 MgCl₂, 10 glucose and 10 HEPES at pH 7.3. Cells were loadedwith 4 μM Fura2-AM for 40 minutes at 37° C. in standard extracellularsolution. Intracellular free Ca2+ ([Ca2+]i) concentrations calculatedusing the Grynkiewicz equation, [Ca2+]i=Kd×□×(R−Rmin)/(Rmax−R) (52).Results are shown in FIG. 15.

RESULTS

As seen in Table 3, each cell line has a distinct cell morphology,indicating that they represent unique cell types demonstrating thepotential diversity from the heterogeneous cells, although all of thecells have some common characteristics, such as overall neuronalphenotype and the appearance of neurites (FIG. 19A). Using electronmicroscopy it was found that the cells exhibit dense core granules,indicative of secretory neurons, and from cell-cell contacts, with whatappears to be the presence of dense material at the contact point,indicative of what has been seen previously at a synaptic cleft (53).(FIG. 19B). The lines are stable and maintain expression of SV40T-antigen and NSE, but do not express GFAP (FIG. 19B). For instance, theN-38 cell line has been passaged to P-44 and maintains SV40 T-antigenexpression after 2 years of continuous growth. Further, all cell linesexpress syntaxin-1, a SNARE protein complex member localized to thepresynaptic plasma membrane that has an indispensable role inneurosecretion (54). These immortalized, clonal cell lines provide validmodel systems for molecular and biochemical investigations on theregulation of specific hormones, characteristics of their respectivesecretory neuronal population and an unlimited source of homogenous cellmaterial and of the neuropeptide itself. The neuropeptides can be usedin the treatment of related neurological conditions.

Expression profiles of the hypothalamic cell lines characterized to dateare described in Table 3. Known hypothalamic markers were examined withparticular attention to neuropeptides linked to energy homeostasis,specific releasing hormones, and enzymes responsible forneurotransmitter synthesis. A wide variety of neuronal phenotypes weregenerated, often with co-expression of multiple markers, confirming whathas been detected in vivo through mainly immunocytochemical methodology(55). The neurons expressing peptides linked to energy homeostasisexpressed peptide profiles consistent with those reported byimmunochemistry or in situ hybridization (4).

For instance all neuropeptide Y (NPY)-expressing neurons, thought tohave orexigenic properties in feeding behaviour, also expressedagouti-related peptide (AgRP), but not proopiomelanocortin (POMC), aprecursor to the anorexigenic neuropeptide alpha-melancortin-stimulatinghormone (56, 57). The successful generation of cell models from thehypothalamus that were not previously available allows the study oftranscriptional regulation of many genes associated with complexneuroendocrine pathways. Specifically, very little is known of thecontrol of melanin-concentrating hormone, urocortin, neurotensin,proglucagon, and growth hormone-releasing hormone in the hypothalamusthe cell lines provided by this invention are the first available tostudy the regulation of these genes. Importantly, many of the linesexpressing peptides associated with energy homeostasis also express thelong form of the leptin receptor, ObR_(b) and suppressor of cytokinesignaling (SOCS), a downstream effector molecule of ObR_(b) (58). As anexample, the N-38 cell line expresses NPY, AgRP and ObR_(b) but not POMC(FIG. 19D). Interestingly, although not expected, all of the cell linesepressed AgRP and GnRH, indicating a potential role for these petides indevelopment. Although a number of cell lines expressed POMC, expressionof cocaine and amphetimine regulated transcript (CART) was not detectedin any of the cell lines, which may suggest a limited window ofexpression of this peptide during development. TABLE 1 Cell Line NamesCELL LINE COMMON NAME eDAY N-1 N-15-1, #1 15 N-2 N-15-1, #2 15 N-3N-15-1, #3 15 N-4 N-15-1, #4 15 N-5 N-15-1, #5 15 N-6 N-15-1, #6 15 N-7N-15-1, #7 15 N-8 N-15-1, #8 15 N-11 N-15-1, #11 15 N-15 N-15-1, #15 15N-17 N-15-1, #17 15 N-18 N-15-1, #18 15 N-19 N-18-1, #6 18 N-22 N-18-1,#11 18 N-20 N-15-14, #20 15 N-25 N-15-14, #25 15 N-29 N-15-14, #29 15N-36 N-15-14, #36 15 N-37 N-17, #2517-6 17 N-38 N-17, #37-1 (NPY-17) 17N-39 N-17, #2517-3 17 N-40 N-17, #1 17 N-41 N-17, #2 17 N-42 N-17, #3 17N-43 N-17, #4 17 N-44 N-17, #6 17 N-45 N-17, #7 17 N-46 N-17, #8 17 N-47N-17, #9 17

TABLE 2 Amplification details used in the RT-PCR analysis of geneexpression. ANNEALING Amplicon POSITIVE CROSS GENE PRIMER SEQUENCE (35cycles) (bp) CONTROL INTRON T-Antigen (T-Ag) S: agaggaatctttgcsgctaa 60433 GT 1-7 yes AS: ctaaacacagcatgactcaa Neuron-Specific Enolase (NSE) S:ctgatgctggagttggatg 60 391 GT 1-7 yes AS: cttcgctgttctccaggatat GlialFibrillary Acidic S: ctgaggctggaggcagagaac 57 621 Hypothalamus yesProtein (GFAP) : AS: cctgtaggtggcgatctcgat Neuropeptide Y (NPY) S:taggtaacaagcgaatgggg 60 282 Hypothalamus yes AS: acatggaagggtcttcaagcIP: gctctgcgacactacatcaa Neurotensin (NT) S: staggaatgaacttcagctg 60 498Hypothalamus yes AS: gtaggaggccctcttgagtat Proglucagon (proGlu) S:ttcaccagcgactacagcaaa 60 351 Hypothalamus yes AS: ggtttgaatcsgccagttgatIP: agtgatgtgagttcttacttg Tyrosine Hydroxylase (TH) S:cagctggaggatgtgtctca 60 140 Hypothalamus yes AS: ggcatgacggstgtactgtgIP: cggtctactgtctgcccgtg Growth Hormone Releasing S:ttgtgatcctcatcctcaccag 60 128 Hypothalamus yes Hormone AS:atcactttccgggcatacag (GHRH) IP: atgccatcttcaccaccaacCorticotropin-Releasing S1: attctgatccgcatgggtgaagaatacttc 60 162Hypothalamus no Hormone (CRH) AS1: taattaggggtatataggctctctccctg IP:tgcagaatcgttttggccaagcgcaacatt Galanin (Gal) S: catgccattgacaaccacag 55320 Hypothalamus yes AS: ggattggcttgaggagttgg (30 cycles) IP:atgtgcccctgcctgagagc Proopiomelanocortin (POMC) S: tagatgtgtggagctggtgc60 149 Hypothalamus yes AS: cagtcaggggctgttcatct IP:aacctgctggcttgcatccg Neurofilament protein (NF) S:cgaagagcgagatggccaggtac yes AS: cactctgcaagcaaacagatact Agouti RelatedPeptide (AgRP) S: agggcatcagaaggcctgaccagg 60 252 Hypothalamus yes AS:ttgaagaagcggcagtagcacgt IP: atccacagaaccgcgagtct Cocaine and AmphetamineS: agctcccgcctgcggctgct 60 299 Hypothalamus yes Regulated Transcript(CART) AS: cagtcacacagcttcccgatcc IP: tgttgcagatcgaagcgttg Urocortin(Ucn) S: gcgtcttcagcccgtccccggggacagagt 60 455 Hypothalamus yes AS:ccgatcacttgcccaccgaatcgaatatg IP: gctacgctcctggtggcgttgctgctcttgIP:-intron: tggaactggactggcacagc Arginine Vasopressin (Avp) S:gccgcgggcatctgctgcagcgatg 60 223 Hypothalamus yes AS:tcagtagacccggggcttggcagaa IP: agcaacgccacacagctggac ThyrotropinReleasing S: ggaccttggctgatgatggct 60 592 Hypothalamus yes Hormone (TRH)AS: tcaggcattaagccaccctcc IP: aagacgttgaagccgaagag Suppressor ofCytokine S: tccacgctggctccgtgcg 57 603 Hypothalamus no Signaling (SOCS3)AS: gctccttaaagtggagcatca Melanin-concentrating S:ttaatgctggctttttctttgttt Hormone (MCH) AS: accgctctcgtcgtttttgta 50 185Hypothalamus yes IP: gcttccaagtccataaggaa Tryptophan Hydroxylase (TPH)S: cattcctccgaaagagggagagtgact 60 254 Hypothalamus AS:agctgatcgggcgagtccaccgaga Oxytosin (OXT) S: ccagtctcgcttgctacctg 55 310Hypothalamus yes AS: gggctgcagcagatgcctgt IP: cgctgcttcggaccaagcatGonadotropin-Releasing S: actgtgtgtttggaaggctgc 62 162 GT 1-7 yesHormone (GnRH) AS: ttccagagctcctcgcagatc (30 cycles) IP:actgtccactggccccgttc Antrogen Receptor (AR) S: acgtcctggaagccattgagcc 60560 GT1-7 yes AS: cttggtgagectggtggaagcgc Estrogen Receptor α(ERα) S:gaattcaattctgacaatcgacgccag 57 344 GT1-7 yes AS:gaattcgtgcttcaacattctccctcc Estrogen Receptor ⊕(ER⊕) ₁:gaattctagccacccactgccaatcat 59 407/332 GT1-7 yes AS₁:gaattccacacctttctctcctggatg Leptin Receptor (Ob-Rb) S:atgacgcagtgtactgctg 60 356 Hypothalamus yes AS: gtggcgagtcaagtgaacct IP:tgtgcagctgaggtatcaca Glucagon-like-peptide 2 S: ccccaaccaagcccggcagtg 60187 Hypothalamus no Receptor (Glp2R) AS: tcttacttgcttgatggaaacc IP:cctgcggcccaggggcctgag Aromatase (Arom) S: ggattggaagtgcctgcaactact 60400 Hypothalamus AS: gagcatgttagaggtgtccagcat IP: cggcatgcatgagaacggcaOrexin (OX) S: cctgagctccaggcaccatgaact AS: tggttaccgttggcctgaaggagg 60232 Hypothalamus yes IP: cagaagacgtgttcctgccg

TABLE 3 Cell Lines Screening N- N- N- N- N- N- N- N- N- N- N- N- MARKERN-1 N-2 N-3 4 N-6 N-7 8 11 19 22 20 25 29 36 37 38 39Tantigen + + + + + + + + + + + + + + + + +NSE + + + + + + + + + + + + + + + + GFAP − − − − − − − − − − − − − − N/A− NT − − − − − − − − − − − − − + − − +ERα + + + + + + + + + + + + + + + + ERβ + − + + + + + + + + + + + + + +Tph − − − − − + − − N/A + + w, − + strong w but RT- weaker Socs-3 +− + + − + + + N/A + + + + + + + + AR − N/A + w + − w − N/A − N/A N/A N/AN/A N/A + − G2R − − w − − + − − + + − − + − − − CRF − − − − − − − − − −− − − − − − w GnRH + + + + + + + + + + + + + + w POMC − − w + − −w + + + + − + − + − Gal + − + − − + + w − − w w w + − − − Lep + + w w +− + + + + − − + w w w + Receptor Agrp + + + + + + + + + + + + + + + + +Cart − − − − − − − − − − − − − − − − − NPY w − − − − − − w − − − − + w− + − proGlu − − − − w − + − − w − − w − + − w TH − + w + + + + + + − +w + + + − GHRN w w + − + − w w + + − w + + + +Avp + + + + + + + + + + + + w + w w proTRH − − − − − − − − − − − − − − −− − Ucn + − w w + − − + − − + + − + − − + MCH N/A N/A + + N/A + N/A N/AN/A N/A N/A N/A + w w + + orexin − − − − − − − − N/A − − − − − − − − DAT− − − − − strong − + N/A − − w w − − − CRFR1 − N/A − − bigger − − − N/A− bigger − − − N/A − size size CRFR2 − N/A − − − − − − N/A − − − − − N/A− Aromatase + N/A − − − − − + N/A − − − − N/A + strong GnRH − N/A − − −− − − N/A − − − − N/A − − Receptor Insulin + + + + + + + +N/A + + + + + + + + receptor Oxytocin + N/A − a bit? w + + + N/A + + + +N/A + + Ten M-1 − − − − − − − − − − − − − − − − Ten M-2 − − − − − − − −− − − − − − − − Ten M-4 + + + + + − + + + − − − + + + − GHS-R N/A N/AN/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A − + Leptin som NTR −− N/A N/A − + − + N/A w N/A N/A N/A N/A w − w mc3R mc4R N/A N/A N/A N/AN/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A − + NPY-Y1 NPY-Y2 CRLR N/AN/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A − + Ghrelin + −N/A N/A − + + − N/A + N/A N/A N/A N/A + + + Ghrelin − − N/A N/A + + + −N/A − N/A N/A N/A N/A + − + variant N- N- N- N- N- N- N- N- N- N- N- N-N- N- MARKER 20/1 20/2 25/2 25/3 25/4 29/1 29/2 29/3 29/4 N-36/1 36/2 4041 42 43 N-44 N-45 N-46 N-47Tantigen + + + + + + + + + + + + + + + + + + + NSE GFAP NT + + + −− + + + + strong + + + + + + + + + ERα ERβ Tph Socs-3 + + + +N/A + + + + + + + + + + + + + + AR + + w − − w − w − − + + + + + + + + +G2R CRF − − − − − − − − − − − − − − − + − − − GnRH POMC Gal + − − − − −− − − + − + + + + + + + + Lep Receptor + + + + − + + + + + + + + + + +w + + Agrp + + + + + + + + + + + + + + + + + + + Cart − − − − − − − − −− − − − − − + − − − NPY − − + w − w − − + − − + + + + + + + + proGlu − +− − − − − − − − − + + − − − − − − TH GHRH Avp proTRH − − − − − − − − − −− − − − − − − − − Ucn + + + + w + + + + + + + + + + w w + + MCH + + + +w + + + + + + + + + + + + + + orexin − + − − − − − − − − − − − − − − − −− DAT CRFR1 CRFR2 Aromatose GnRH Receptor Insulinreceptor + + + + + + + + + + + + + + + + + + + Oxytocin New-1 New-2New-4 GHS-R − − − − N/A + + + − − − − − − − + − + − Leptin som NTR + w −− N/A w − − − + w − − − w − − − − mc3R mc4R − − + + N/A + − − −− + + + + + ? + + + NPY-Y1 NPY-Y2 CRLR + + − − N/A + − −− + + + + + + + + + + Ghrelin + + + − N/A + + + + + + + + + + + + + +Ghrelin variant − + − + N/A − + − + w − + + − − + − + −

TABLE 4 Genes Regulated by TCAP-1 at 16 hours Affimetrix Acc No. FoldCluster Gene Probe No. GB Function change Growth/ GAS5 98530 AI849615Growth arrest specific transcript 0.46 Differentiation SDPR 160373AI839175 Serum deprivation response protein 0.57 PPAN 160802 AA674812Peter Pan homologue 0.62 CD95 102921 M83649 Fas antigen 0.61 CRD-BP102627 AF061569 CRD-binding protein 0.59 SSG1 160298 AW122012 Steroidsensitive gene 1 0.62 DLP1/2 97353 AI837497 DAB2 interacting protein0.68 GBP3 103202 AW047476 Guanylate binding protein 0.63 P202 161173AV229143 202 interferon activatable protein 0.61 CAI1 103441 AI94248Casein kinase II 0.61 INI1B 99924 AW121845 Integrase interacting protein1B 0.48 MMP1 100484 X66473 Matrix metalloproteinase 1 0.55 MMP10 94724Y13185 Matrix metalloproteinase 10 0.59 PTK7 92325 AI326889 Receptorprotein tyrosine kinase 1.53 P204 98466 M31419 Interferon activatableprotein 1.85 MKI67 161931 AV309347 Cell cycle protein regulator 1.70MOP3 102382 AB014494 Circadian rhythm regulator 1.57 ST7 160591 AI504013Suppressor of tumourigenicity 1.97 GDAP10 94192 Y17860 Gangliosideinduced diff. protein 10 1.62 Signalling/ ERK1 101834 ZI4249 Mitogenactivated protein kinase 0.64 Communication ALK3 92767 DI6250 Bonemorphogenic protein receptor 0.60 BMP4 93456 L47480 Bone morphogenicprotein-4 0.52 IL1R 93914 M20658 Interleukin 1 receptor 0.60 GR 98818X04435 Glucocorticoid receptor 0.66 BARK1 104270 AA982714 β adrenorgicreceptor kinase 1 0.61 CAMIII 92631 M19380 Calmodulin III 0.53 PCDHγ160976 AA222943 protocadherin γ 0.42 AKAP95 95001 AB028920 A kinaseanchor protein 95 0.60 TTF-1IP 161019 W41560 TTF-1 interacting peptide0.50 CREMβ1 100533 M60285 cAMP-responsive element modulator 1.61 AKAP8161088 AV171460 A kinase anchor protein 8 1.58 PDE6A 100696 X60664 cGMPPhosphodiesterase α 1.68 INOS 104420 U43428 Inducible nitric oxidesynthetase 1.50 FNBX 92754 D49920 Ferredoxin-NADP reductase 1.61 SLC6A4161695 AV230927 Serotonin transporter 1.53 CLCN3 94465 AF029347 Chloridechannel protein 3 1.66 Processing ARF1 95156 AI1853873 ADP ribosylationfactor 1 0.63 CLM2-B 93492 AB013469 Cytohesin-2 0.63 YIPID 99675AI839766 Rab-mediated membrane transport 1.88 RAB10 160149 AI841543 Rasoncogene homologue 1.62 GP25L2 100074 AW046723 gp25L brings cargoforward from ER 1.53 AP4S1 104561 AI847561 Adaptor related proteincomplex 1.52

The change in expression levels is indicated relative to the untreatedcontrol cell for the same time period of 16 hours. Values>1.5 fold or<0.70 fold were considered significant.

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1. An immortalized cell line of murine hypothalmic neuronal cellscomprising a gene encoding polyoma virus large T antigen operably linkedto a promoter and expressing a marker selected from the group consistingof neuropeptide Y, gonadotropin-releasing hormone, growth-hormonereleasing hormone (GHRH), TenM 1, 2, 3, 4, arginine vasopressin (AVP),thyrotropin-releasing hormone (TRH), SOCS-3, urocortin,melanocortin-concentrating hormone (MCH), orexin, dopamine transporter,corticotrophin-releasing factor (CRF), gonadotropin releasing hormonereceptor, tryptophan hydroxylase, tyrosine hydroxylase, galanin,proopiomelanocortin (POMC), proglucagon, neurotensin, somatostatin,agouti-related protein, cocaine and amphetamine-regulated transcript(CART), leptin, oxytocin, corticotrophin-releasing factor receptor 1 and2, aromatase, ghrelin, growth hormone secratogue receptor, androgenreceptor, estrogen receptor α, estrogen receptor β, leptin receptor,melanocortin-concentrating hormone receptor 3 and 4, neuropeptide Yreceptor Y1, neuropeptide Y receptor Y2, calcitonin receptor likereceptor, glucagon-like peptide 1 receptor, glucagon-like peptide 2receptor (Glp-2 receptor), and neurotensin receptor.
 2. A mixed cellpopulation comprising the immortalized cell line of claim
 1. 3. Theimmortalized cell line of claim 1 wherein the marker is Glp-2 receptor.4. The immortalized cell line of claim 1 wherein the marker isneurotensin.
 5. The immortalized cell line of claim 1 wherein the markeris proopiomelanocortin (POMC).
 6. The immortalized cell line of claim 1wherein the marker is neuropeptide Y (NPY).
 7. The immortalized cellline of claim 1 wherein the marker is proglucagon.
 8. The immortalizedcell line of claim 1 wherein the marker is growth-hormone releasinghormone.
 9. The immortalized cell line of claim 1 wherein the marker isurocortin.
 10. The immortalized cell line of claim 1 wherein the markeris melanocortin-concentrating hormone.
 11. The immortalized cell line ofclaim 1 wherein the marker is TenM
 4. 12. The immortalized cell line ofclaim 1 wherein the marker is growth hormone secratogue receptor. 13.The immortalized cell line of claim 1 wherein the marker is ghrelin. 14.An immortalized cell line of claim 1 prepared by the method comprising:(i) preparing a culture of embryonic hypothalamic cells; (ii) infectingsaid culture with a retrovirus encoding a viral oncogene, operablylinked to a promoter and a selectable marker; (iii) isolatingtransfected cells from non-transfected cells to obtain a culture ofimmortalized hypothalamic cells; (iv) subcloning said immortalized cellsinto sub-cloned populations; (v) screening said subcloned populationsfor expression of specific neuronal markers; and (vi) selecting andfurther cloning a specific population.
 15. A method of obtaining aneuropeptide comprising, culturing the cell line of claim 1 that isknown to express said neuropeptide and isolating the expressedneuropeptide.
 16. A method for identifying a modulator of a neuropeptidecomprising: (i) providing a cell line as defined in claim 1; (ii)incubating the cell line in the presence of the candidate modulator; and(iii) determining the biological effect of said candidate modulator,wherein said candidate is a modulator if it modulates the neuropeptideexpression and/or activity.
 17. The method of claim 15 wherein saideffect of said candidate modulator can be determined by one of thefollowing methods: (a) monitoring effects on neuropeptide expression;(b) incubating the said cell line with a substrate of a neuropeptide andmonitoring the effect on substrate metabolites; (c) binding assays; or(d) proteomic profiling in the presence and absence of the saidcandidate modulator.
 18. An immortalized cell line of murine hypothalmicneuronal cells that is responsive to a teneurin C-terminal-associatedpeptide [TCAP].
 19. The cell line of claim 16 wherein the teneurinC-terminal-associated peptide is selected from the group consisting ofSEQ ID NOs 1-9.
 20. The cell line of claim 16 wherein the teneurinC-terminal-associated peptide is murine TCAP-1 or TCAP-3.
 21. The cellline of claim 16 wherein the cell line is selected from the groupconsisting of N-7, N-22, N-29 and N-38.